Compositions of pegylated soluble tumor necrosis factor receptors and methods of preparing

ABSTRACT

The present invention provides for improved compositions comprising a PEGsTNF-R1 which, in addition to having useful higher concentrations, demonstrate decreased viscosity (&lt;400 cP) and improved stability.

This application is a continuation of U.S. nonprovisional application14/613,269, filed Feb. 3, 2015, pending, which is a continuation of U.S.nonprovisional application Ser. No. 12/432,452, filed Apr. 29, 2009,abandoned, which is a continuation of Ser. No. 10/461,839, filed Jun.12, 2003, now U.S. Pat. No. 7,700,722, which is a continuation in partof U.S. nonprovisional application Ser. No. 10/177,566, filed Jun. 20,2002, abandoned, all of which are hereby incorporated by reference.

The present application incorporates by reference in its entirety allsubject matter contained in the attached sequence listing which is intxt format and is identified by the name of the file,40030D_SeqListing.txt, created on Feb. 3, 2017, the size of which fileis 1,401 bytes.

BACKGROUND OF THE INVENTION

Inflammation is the body's defense reaction to injuries such as thosecaused by mechanical damage, infection or antigenic stimulation. Aninflammatory reaction may be expressed pathologically when inflammationis induced by an inappropriate stimulus such as an autoantigen, isexpressed in an exaggerated manner, or persists well after the removalof the injurious agents. Such inflammatory reaction may include theproduction of certain cytokines.

While the etiology of inflammation is poorly understood, considerableinformation has recently been gained regarding the molecular aspects ofinflammation. This research has led to identification of certaincytokines which are believed to figure prominently in the mediation ofinflammation. Cytokines are extracellular proteins that modify thebehavior of cells, particularly those cells that are in the immediatearea of cytokine synthesis and release. Tumor necrosis factors (TNFs)are a class of cytokines produced by numerous cell types, includingmonocytes and macrophages.

At least two TNFs have been previously described, specifically TNF alpha(TNF-α) and TNF beta (TNF-β or lymphotoxin), and each is active as atrimeric molecule and is believed to initiate cellular signaling bycrosslinking receptors; Engelmann et al., J. Biol. Chem.,265:14497-14504 (1990).

Several lines of evidence implicate TNF-α and TNF-β as majorinflammatory cytokines. These known TNFs have important physiologicaleffects on a number of different target cells which are involved ininflammatory responses to a variety of stimuli such as infection andinjury. The proteins cause both fibroblasts and synovial cells tosecrete latent collagenase and prostaglandin E₂ and cause osteocytecells to stimulate bone resorption. These proteins increase the surfaceadhesive properties of endothelial cells for neutrophils. They alsocause endothelial cells to secrete coagulant activity and reduce theirability to lyse clots. In addition they redirect the activity ofadipocytes away from the storage of lipids by inhibiting expression ofthe enzyme lipoprotein lipase. TNFs also cause hepatocytes to synthesizea class of proteins known as “acute phase reactants,” which act on thehypothalamus as pyrogens; Selby et al., Lancet, 1(8583):483 (1988);Starnes, Jr. et al., J. Clin. Invest., 82:1321 (1988); Oliff et al.,Cell, 50:555 (1987); and Waage et al., Lancet, 1(8529):355 (1987).Additionally, preclinical results with various predictive animal modelsof inflammation, including rheumatoid arthritis, have suggested thatinhibition of TNF can have a major impact on disease progression andseverity; Dayer et al., European Cytokine Network, 5(6):563-571 (1994)and Feldmann et al., Annals Of The New York Academy Of Sciences,66:272-278 (1995). Moreover, recent preliminary human clinical trials inrheumatoid arthritis with inhibitors of TNF have shown promisingresults; Rankin et al., British Journal Of Rheumatology, 3(4):4334-4342(1995); Elliott et al., Lancet, 344:1105-1110 (1995); Tak et al.,Arthritis and Rheumatism, 39:1077-1081 (1996); and Paleolog et al.,Arthritis and Rheumatism, 39:1082-1091 (1996).

Protein inhibitors of TNF are disclosed in the art. EP 308378 reportsthat a protein derived from the urine of fever patients has a TNFinhibiting activity. The effect of this protein is presumably due to acompetitive mechanism at the level of the receptors. EP 308378 disclosesa protein sufficiently pure to be characterized by its N-terminus. Thereference, however, does not teach any DNA sequence or arecombinantly-produced TNF inhibitor.

Recombinantly-produced TNF inhibitors have also been taught in the art.For example, EP 393438 and EP 422339 teach the amino acid and nucleicacid sequences of a mature, recombinant human “30 kDa TNF inhibitor”(also known as a p55 receptor and as sTNFR-I) and a mature, recombinanthuman “40 kDa inhibitor” (also known as a p75 receptor and as sTNFR-II)as well as modified forms thereof, e.g., fragments, functionalderivatives and variants. EP 393438 and EP 422339 also disclose methodsfor isolating the genes responsible for coding the inhibitors, cloningthe gene in suitable vectors and cell types, and expressing the gene toproduce the inhibitors. Mature recombinant human 30 kDa TNF inhibitorand mature recombinant human 40 kDa TNF inhibitor have been demonstratedto be capable of inhibiting TNF (EP 393438, EP 422339, PCT WO 92/16221and PCT WO 95/34326).

sTNFR-I and sTNFR-II are members of the nerve growth factor/TNF receptorsuperfamily of receptors which includes the nerve growth factor receptor(NGF), the B cell antigen CD40, 4-1BB, the rat T-cell antigen MRC OX40,the Fas antigen, and the CD27 and CD30 antigens; Smith et al., Science,248:1019-1023 (1990). The most conserved feature amongst this group ofcell surface receptors is the cysteine-rich extracellular ligand bindingdomain, which can be divided into four repeating motifs of about fortyamino acids and which contains 4-6 cysteine residues at positions whichare well conserved; Smith et al., supra.

EP 393438 further teaches a 40 kDa TNF inhibitor 451 and a 40 kDa TNFinhibitor 453, which are truncated versions of the full-lengthrecombinant 40 kDa TNF inhibitor protein wherein 51 or 53 amino acidresidues, respectively, at the carboxyl terminus of the mature proteinare removed. Accordingly, a skilled artisan would appreciate that thefourth domain of each of the 30 kDa TNF inhibitor and the 40 kDainhibitor is not necessary for TNF inhibition. In fact, various groupshave confirmed this understanding. Domain-deletion derivatives of the 30kDa and 40 kDa TNF inhibitors have been generated, and those derivativeswithout the fourth domain retain full TNF binding activity while thosederivatives without the first, second or third domain, respectively, donot retain TNF binding activity; Corcoran et al., Eur. J. Biochem.,223:831-840 (1994); Chih-Hsueh et al., The Journal of BiologicalChemistry, 270(6):2874-2878 (1995); and Scallon et al., Cytokine,7(8):759-770 (1995).

PCT WO US97/12244 describes functionally active truncated forms ofsTNFR-I and sTNFR-II (referred to as “truncated sTNFR(s)). The truncatedsTNFRs are modified forms of sTNFR-I and sTNFR-II which do not containthe fourth domain (amino acid residues Thr¹²⁷-Asn¹⁶¹ of sTNFR-I andamino acid residues Pro¹⁴¹-Thr¹⁷⁹ of sTNFR-II); a portion of the thirddomain (amino acid residues Asn¹¹¹-Cys¹²⁶ of sTNFR-I and amino acidresidues Pro¹²³-Lys¹⁴⁰ of sTNFR-II); and, optionally, which do notcontain a portion of the first domain (amino acid residues Asp¹-Cys¹⁹ ofsTNFR-I and amino acid residues Leu¹-Cys³² of sTNFR-II).

PEG-rmet-Hu-sTNF-R1 (PEGsTNF-R1) as described herein is a recombinantform of a functionally active truncated form of sTNFR-I and sTNFR-IIwhich has been PEGylated at the N-terminus with, e.g., a 30 kDapolyethylene glycol molecule. In our preliminary studies with PEGsTNF-R1it was found that as the PEGsTNF-R1 is concentrated, the viscosity ofthe solution increases exponentially. Large scale methods traditionallyused for concentrating proteins are known to be unsatisfactory whenworking with such viscous solutions, and the increased viscosity mayprevent concentrating the protein to high concentrations withoutdamaging the final product. Because there may be instances in acommercial setting where it will be necessary to have the protein at ahigher concentration (e.g., >45 mg/ml) in order to deliver to requiredtherapeutic dose, there is a need to develop formulations which obtainsuch concentrations and with acceptable low viscosities (e.g., <400 cP)to allow for the use of the various delivery devices necessary fordelivery of the therapeutic dose. For example, in order to deliver therequired therapeutic dose of a PEGsTNF-R1 formulation wherein thePEGsTNF-R1 concentration is >45 mg/ml, and using a commerciallyavailable autoinjector and pre-filled syringe as the delivery device,the formulation should have a viscosity of <400 cP. Above thisviscosity, the strong possibility exists for the device or container tofail. The present invention provides for PEGsTNF-R1 formulations havingsuch concentrations and low viscosities, thereby allowing for use ofdelivery devices which are more convenient and patient-friendly.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a new and improvedformulation of PEGsTNF-R1, wherein said formulation has a concentrationof at least 45 mg/ml without apparent damage to the protein, and withdecreased viscosity (<400 cP) and improved stability.

Also provided are methods of preparing such formulations, said methodsbeing capable of being scaled up to accommodate a commercial setting.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptide is defined herein as natural, synthetic, and recombinantproteins or peptides having more than about 10 amino acids, and having adesired biological activity. Proteins contemplated for use herein wouldinclude but are not limited to interferon consensus (see, U.S. Pat. Nos.5,372,808, 5,541,293 4,897,471, and 4,695,623 hereby incorporated byreference including drawings), granulocyte-colony stimulating factors(see, U.S. Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCTPublication No. 94/17185, hereby incorporated by reference includingdrawings), interleukins (see, U.S. Pat. No. 5,075,222, herebyincorporated by reference including drawings), erythropoietins (see,U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080hereby incorporated by reference including drawings), stem cell factor(PCT Publication Nos. 91/05795, 92/17505 and 95/17206, herebyincorporated by reference including drawings), osteoprotegerin (PCTPublication No. 97/23614, hereby incorporated by reference includingdrawings), novel erythropoiesis stimulating protein (NESP) (PCTPublication No. 94/09257, hereby incorporated by reference includingdrawings), leptin (OB protein)(see PCT publication Nos. 96/40912,96/05309, 97/00128, 97/01010 and 97/06816 hereby incorporated byreference including figures), megakaryocyte growth differentiationfactor (see, PCT Publication No. 95/26746 hereby incorporated byreference including figures), tumor necrosis factor inhibitors, e.g.,sTNF-R1 (see, PCT WO US97/12244 hereby incorporated by referenceincluding figures), interleukin-1 receptor antagonist (IL-1ra), brainderived neurotrophic factor (BDNF), glial derived neurotrophic factor(GDNF), keratinocyte growth factor (KGF) and thrombopoietin. The termproteins, as used herein, includes peptides, polypeptides, consensusmolecules, analogs, derivatives or combinations thereof.

In general, the sTNFRs contemplated for use in the present invention arethose described in PCT WO US97/12244, and references cited therein.Specifically, the sTNFRs will be the truncated sTNFRs described therein.The truncated sTNFRs may advantageously be produced via recombinanttechniques in bacterial, mammalian or insect cell systems and may beeither a glycosylated or non-glycosylated forms of the protein.Alternatively, truncated sTNFRs may be chemically synthesized. Currentlypreferred production methods are described in PCT WO US97/12244.

Truncated sTNFRs each may typically be isolated and purified to besubstantially free from the presence of other proteinaceous materials(i.e., non-truncated sTNFRs). Preferably, a truncated sTNFR is about 80%free of other proteins which may be present due to the productiontechnique used in the manufacture of the truncated sTNFR. Morepreferably a truncated sTNFR is about 90% free of other proteins,particularly preferably about 95% free of other proteins, and mostpreferably about >98% free of other proteins. Currently preferredisolation and purification methods are described in PCT WO US97/12244.It will be appreciated, however, that the desired protein may becombined with other active ingredients, chemical compositions and/orsuitable pharmaceutical formulation materials prior to administration.

In one aspect of the present invention, the truncated sTNFRs will bederivatized by attaching the truncated sTNFRs to a water solublepolymer. For example, the truncated sTNFRs will be conjugated to one ormore polyethylene glycol molecules in order to improve pharmacokineticperformance by increasing the molecule's apparent molecular weight.

Water soluble polymers are desirable because the protein to which eachis attached will not precipitate in an aqueous environment, such as aphysiological environment. Preferably, the polymer will bepharmaceutically acceptable for the preparation of a therapeutic productor composition. One skilled in the art will be able to select thedesired polymer based on such considerations as whether thepolymer/protein conjugate will be used therapeutically and, if so, thedesired dosage, circulation time and resistance to proteolysis.

Suitable, clinically acceptable, water soluble polymers include, but arenot limited to, polyethylene glycol (PEG), polyethylene glycolpropionaldehyde, copolymers of ethylene glycol/propylene glycol,monomethoxy-polyethylene glycol, carboxymethylcellulose, polyacetals,polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (β-aminoacids) (either homopolymers or random copolymers), poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers (PPG)and other polyakylene oxides, polypropylene oxide/ethylene oxidecopolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylatedglucose, colonic acids or other carbohydrate polymers, Ficoll or dextranand mixtures thereof.

As used herein, polyethylene glycol is meant to encompass any of theforms that have been used to derivatize other proteins, such asmono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water.

The water soluble polymers each may be of any molecular weight and maybe branched or unbranched. The water soluble polymers each typicallyhave an average molecular weight of between about 2 kDa to about 100 kDa(the term “about” indicating that in preparations of a water solublepolymer, some molecules will weigh more, some less, than the statedmolecular weight). The average molecular weight of each water solublepolymer preferably is between about 5 kDa and about 50 kDa, morepreferably between about 12 kDa and about 40 kDa and most preferablybetween about 20 kDa and about 35 kDa.

Generally, the higher the molecular weight or the more branches, thehigher the polymer:protein ratio. Other sizes may be used, depending onthe desired therapeutic profile (e.g., the duration of sustainedrelease; the effects, if any, on biological activity; the ease inhandling; the degree or lack of antigenicity and other known effects ofa water soluble polymer on a therapeutic protein).

The water soluble polymers each should be attached to the protein withconsideration of effects on functional or antigenic domains of theprotein. In general, chemical derivatization may be performed under anysuitable condition used to react a protein with an activated polymermolecule. Activating groups which can be used to link the water solublepolymer to one or more proteins include the following: sulfone,maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxiraneand 5-pyridyl.

The water soluble polymers each are generally attached to the protein atthe α- or ε-amino groups of amino acids or a reactive thiol group, butit is also contemplated that a water soluble group could be attached toany reactive group of the protein which is sufficiently reactive tobecome attached to a water soluble group under suitable reactionconditions. Thus, a water soluble polymer may be covalently bound to aprotein via a reactive group, such as a free amino or carboxyl group.The amino acid residues having a free amino group may include lysineresidues and the N-terminal amino acid residue. Those having a freecarboxyl group may include aspartic acid residues, glutamic acidresidues and the C-terminal amino acid residue. Those having a reactivethiol group include cysteine residues.

Methods for preparing proteins conjugated with water soluble polymerswill each generally comprise the steps of (a) reacting a protein with awater soluble polymer under conditions whereby the protein becomesattached to one or more water soluble polymers and (b) obtaining thereaction product. Reaction conditions for each conjugation may beselected from any of those known in the art or those subsequentlydeveloped, but should be selected to avoid or limit exposure to reactionconditions such as temperatures, solvents and pH levels that wouldinactivate the protein to be modified. In general, the optimal reactionconditions for the reactions will be determined case-by-case based onknown parameters and the desired result. For example, the larger theratio of water soluble polymer:protein conjugate, the greater thepercentage of conjugated product. The optimum ratio (in terms ofefficiency of reaction in that there is no excess unreacted protein orpolymer) may be determined by factors such as the desired degree ofderivatization (e.g., mono-, di-, tri-, etc.), the molecular weight ofthe polymer selected, whether the polymer is branched or unbranched andthe reaction conditions used. The ratio of water soluble polymer (e.g.,PEG) to protein will generally range from 1:1 to 100:1. One or morepurified conjugates may be prepared from each mixture by standardpurification techniques, including among others, dialysis, salting-out,ultrafiltration, ion-exchange chromatography, gel filtrationchromatography and electrophoresis.

A variety of approaches have been used to attach the polyethylene glycolmolecules to the protein (PEGylation). For example, Royer (U.S. Pat. No.4,002,531) states that reductive alkylation was used for attachment ofpolyethylene glycol molecules to an enzyme. Davis et al. (U.S. Pat. No.4,179,337) disclose PEG:protein conjugates involving, for example,enzymes and insulin. Shaw (U.S. Pat. No. 4,904,584) disclose themodification of the number of lysine residues in proteins for theattachment of polyethylene glycol molecules via reactive amine groups.Hakimi et al. (U.S. Pat. No. 5,834,594) disclose substantiallynon-immunogenic water soluble PEG:protein conjugates, involving forexample, the proteins IL-2, interferon alpha, and IL-1ra. The methods ofHakimi et al. involve the utilization of unique linkers to connect thevarious free amino groups in the protein to PEG. Kinstler et al. (U.S.Pat. Nos. 5,824,784 and 5,985,265) teach methods allowing forselectively N-terminally chemically modified proteins and analogsthereof, including G-CSF and consensus interferon. Importantly, thesemodified proteins have advantages as relates to protein stability, aswell as providing for processing advantages.

The preferred method of the present invention is the selectiveN-terminal chemical modification as described by Kinstler et al. (U.S.Pat. Nos. 5,824,784 and 5,985,265). As taught by Kinstler et al., onemay selectively attach a water soluble polymer to the N-terminus of theprotein by performing the reaction at a pH which allows one to takeadvantage of the pKa differences between the ε-amino group of the lysineresidues and that of the α-amino group of the N-terminal residue of theprotein. By such selective derivatization, attachment of a water solublepolymer to a protein is controlled: the conjugation with the polymertakes place predominantly at the N-terminus of the protein and nosignificant modification of other reactive groups, such as the lysineside chain amino groups, occurs. Using reductive alkylation, the watersoluble polymer may be of the type described above and should have asingle reactive aldehyde for coupling to the protein. Polyethyleneglycol propionaldehyde, containing a single reactive aldehyde, may beused.

By such selective derivatization, attachment of a water soluble polymer(that contains a reactive group such as an aldehyde) to a protein iscontrolled: the conjugation with the polymer takes place predominantlyat the N-terminus of the protein and no significant modification ofother reactive groups, such as the lysine side chain amino groups,occurs. The preparation will typically be greater than 90%monopolymer/protein conjugate, and more typically greater than 95%monopolymer/protein conjugate, with the remainder of observablemolecules being unreacted (i.e., protein lacking the polymer moiety).

A specific embodiment of the present invention is an unbranchedmonomethoxy-polyethylene glycol aldehyde molecule having an averagemolecular weight of either about 20 kDa or about 33 kDa (e.g., between30 kDa and 35 kDa), or a tertiary-butyl polyethylene glycol aldehydehaving an average molecular weight of about 33 kDa (e.g., between 30 kDaand 35 kDa) conjugated via reductive alkylation to a truncated sTNFR,wherein the truncated sTNFR has the amino acid sequence depicted in SEQID NO:1.

The pegylation also may specifically be carried out via water solublepolymers having at least one reactive hydroxy group (e.g. polyethyleneglycol) reacted with a reagent having a reactive carbonyl, nitrile orsulfone group to convert the hydroxyl group into a reactive Michaelacceptor, thereby forming an “activated linker” useful in modifyingvarious proteins to provide improved biologically-active conjugates.“Reactive carbonyl, nitrile or sulfone” means a carbonyl, nitrile orsulfone group to which a two carbon group is bonded having a reactivesite for thiol-specific coupling on the second carbon from the carbonyl,nitrile or sulfone group (WO 92/16221). The activated linkers can bemonofunctional, bifunctional, or multifunctional. Useful reagents havinga reactive sulfone group that can be used in the methods include,without limitation, chlorosulfone, vinylsulfone and divinylsulfone.

PCT International Application No. US96/19459, the disclosure of which ishereby incorporated by reference, teaches methods of makingsulfone-activated linkers by obtaining a compound having a reactivehydroxyl group and converting the hydroxyl group to a reactive Michaelacceptor to form an activated linker, with the use of tetrahydrofuran(THF) as the solvent for the conversion. Also described is a process forpurifying the activated linkers which utilizes hydrophobic interactionchromatography to separate the linkers based on size and end-groupfunctionality.

Pharmaceutical compositions of the present invention will generallyinclude a therapeutically effective amount of a chemically-modifiedderivative of truncated sTNFRs in admixture with a vehicle. The primarysolvent in a vehicle may be either aqueous or non-aqueous in nature. Inaddition, the vehicle may contain other pharmaceutically acceptableexcipients. Excipient is defined herein as a non-therapeutic agent addedto a pharmaceutical composition to provide a desired effect, e.g.stabilization, isotonicity. Common attributes of desirable excipientsare aqueous solubility, non-toxicity, non-reactivity, rapid clearancefrom the body, and the absence of immunogenicity. In addition, theexcipients should be capable of stabilizing the native conformation ofthe protein so as to maintain the efficacy and safety of the drug duringprocessing, storage and administration to the patient.

It is envisioned that the formulations of the present invention willadditionally contain a buffering agent, e.g., alkali salts (sodium orpotassium phosphate or their hydrogen or dihydrogen salts), sodiumcitrate/citric acid, sodium acetate/acetic acid, and any otherpharmaceutically acceptable ph buffering agent known in the art, tomaintain the pH of the solution within a desired range. Mixtures ofthese buffering agents may also be used. The amount of buffering agentuseful in the composition depends largely on the particular buffer usedand the pH of the solution. For example, acetate is a more efficientbuffer at pH 5 than pH 6 so less acetate may be used in a solution at pH5 than at pH 6. The preferred pH of the preferred formulations will bein the range of 4.0 to 5.0, and pH-adjusting agents such as hydrochloricacid, citric acid, sodium hydroxide, or a salt thereof, may also beincluded in order to obtain the desired pH.

The formulations of the present invention may further include one ormore tonicity modifiers to render the solution isotonic with a patient'sblood for injection. Typical tonicity modifiers are well known in theart and include but are not limited to various salts, amino acids orpolysaccharides. Non-limiting examples of suitable amino acids includeglycine. Non-limiting examples of suitable polysaccharides includesucrose, mannitol and sorbitol. It is understood that more than onetonicity modifier may be used at once, for example, sorbitol and glycinecan be used in combination to modify a formulation's tonicity.

It is also envisioned that other anti-oxidants may be included in theformulations of the present invention. Anti-oxidants contemplated foruse in the preparation of the formulations include amino acids such asglycine and lysine, chelating agents such as EDTA and DTPA, andfree-radical scavengers such as sorbitol and mannitol.

Other effective administration forms such as parenteral slow-releaseformulations, inhalant mists, orally-active formulations, orsuppositories are also envisioned. As such, the formulations may alsoinvolve particulate preparations of polymeric compounds such as bulkerosion polymers (e.g., poly(lactic-co-glycolic acid) (PLGA) copolymers,PLGA polymer blends, block copolymers of PEG, and lactic and glycolicacid, poly(cyanoacrylates)); surface erosion polymers (e.g.,poly(anhydrides) and poly(ortho esters)); hydrogel esters (e.g.,pluronic polyols, poly(vinyl alcohol), poly(vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, cellulose, hyaluronic acidderivatives, alginate, collagen, gelatin, albumin, and starches anddextrans) and composition systems thereof; or preparations of liposomesor microspheres. Such formulations may influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thepresent proteins and derivatives. The optimal pharmaceutical formulationfor a desired protein will be determined by one skilled in the artdepending upon the route of administration and desired dosage. Exemplarypharmaceutical formulations are disclosed in Remington's PharmaceuticalSciences, 18th Ed. (1990), Mack Publishing Co., Easton, Pa. 18042, pages1435-1712, the disclosure of which is incorporated herein by reference.

Filtration is a pressure driven separation process that uses membranesto separate components in a liquid or suspension based on their size andcharge differences. Membrane-based Tangential Flow Filtration (TFF) unitoperations are commonly used for clarifying, concentrating, andpurifying proteins. In TFF, the fluid is pumped tangentially along thesurface of the membrane. An applied pressure serves to force a portionof the fluid through the membrane to the filtrate side. The retainedcomponents do not build up at the surface of the membrane, and insteadthey are swept along by the tangential flow. TFF can be furthercategorized base on the size of components being separated. A membranepores size rating, typically given as a micron value, indicates thatparticles larger than the rating will be retained by the membrane. Anominal molecular weight limits (NMWL) is an indication that mostdissolved macromolecules with molecular weights higher than the NMWL andsome with molecular weights lower than the NMWL will be retained by themembrane. A components shape, its ability to deform, and its interactionwith other components in the solution all affect its retention.

Ultrafiltration (UF) is one of the most widely used forms of TFF and isused to separate proteins from buffer components for buffer exchange,desalting, and concentration. Depending on the protein to be retained,membranes NMWLs in the range of 1 kD to 1000 kD are used. The typicalsequences of steps in an ultrafiltration process include cleaning themembranes and the system, testing the integrity and permeability,equilibrating with process buffer, concentrating the sample containingthe product, removing product from system, cleaning the membranes andthe system, testing integrity and permeability, and storing.

The most important factors in design of UF processes include productyield, product quality, process time, and process economics. Yieldlosses in a UF process can be generally be attributed to sieving,solubility limitations, adsorption to the membrane, and unrecoverablevolumetric losses. While several key process parameters such astransmembrane pressure, crossflow rate, and membrane area need to beoptimized, protein concentration is one of the limiting factors indeveloping a TFF step. Since there is the potential for highlyconcentrated areas to exist within the TFF unit as the result of thepolarization, high protein concentration can exceed a solubilitylimitation and increase fouling behavior at membrane surface. Thesignificant increased viscosity (e.g., >500 cP) associated with theconcentration of certain pegylated protein causes process difficultiesin term of maintaining crossflow rate and minimizing heat introduction.The present invention provides an improved ultrafiltration process toconcentrate PEGsTNF-R1 to greater than 45 mg/ml by utilizing a improvedformulation and temperature effect.

The process of lyophilization is very well documented in literature.Lyophilization is the process by which the moisture content of theproduct is reduced by freezing and subsequent sublimation under vacuum.The lyophilization process primarily consists of three stages. The firststage involves freezing the product and creating a frozen matrixsuitable for drying. This step impacts the drying characteristics in thenext two stages. The second stage is primary drying. Primary dryinginvolves the removal of the ice by sublimation by reducing the pressure(to typically around 50-500 μm Hg) of the product's environment whilemaintaining the product temperature at a low, desirable level. The thirdstage in the process is called secondary drying where the bound water isremoved until the residual moisture content reaches below the targetlevel.

Lyophilization improves product stability by (a) maintaining the proteinin an amorphous phase with its stabilizers, (b) immobilizing the proteinin a glassy phase below the glass transition temperature (Tg′) of theformulation, and (c) reducing the residual moisture content to a low,desirable value. Maintaining the protein in an amorphous phase with itsstabilizers helps in protecting the protein. Keeping the dried proteinbelow its glass transition temperature minimizes protein immobility onall practical time-scales and therefore prevents degradation. Reducingthe amount of residual water minimizes all water-catalyzed degradations.

A freeze dryer consists of a chamber with shelves on to which the filledvials are loaded for lyophilization, a condenser for capturing theproduct's sublimed water vapor as ice, a refrigeration system thatfacilitates temperature control, and a vacuum pump which can reduce thechamber pressure to sub-atmospheric values. The chamber pressure ismaintained at its set-point by introducing, in a controlled manner, aninert, dry bleed gas (such as nitrogen) at the front of the chamber. Inmost cases, the chamber is separated from the condenser via a mainvalve. The product is loaded onto the stainless steel shelves, whosetemperature is controlled via a heat-transfer fluid (silicone oil)circulating through the shelves. Temperature of the heat-transfer fluidis controlled via the refrigeration system.

The freezing stage is initiated by cooling the shelves to the desiredfreezing temperature and holding the temperature constant forequilibration. The cooled shelves help freeze the product to the desiredtemperature. Following freezing, the chamber pressure (measured by acapacitance manometer) is reduced to below the saturated vapor pressureof ice at the frozen temperature. This initiates primary drying. Sinceambient pressure is below the saturated vapor pressure at thattemperature, part of the frozen product instantaneously sublimes (thedifference between the vapor pressure of ice and the chamber pressureprovides the driving force for sublimation). Sublimation leads topressure equilibration. However, since the chamber pressure isconstantly maintained below the saturated vapor pressure of ice (at thattemperature), sublimation continues. The sublimed vapors are trapped atthe condenser as ice. Typically, the condenser coil or plates remain atabout −50° C. to −70° C. during the drying process. When all the bulkwater is removed via sublimation, primary drying is complete. At thispoint, there is still some bound water remaining in the product whichcan be removed by desorption at higher temperatures experienced duringsecondary drying. So, typically the shelf-temperature is raised at thisstage and held, until the desired residual moisture is achieved. At thatpoint, secondary drying is also complete, and the vials are stoppered inthe chamber. The chamber is aerated prior to the unloading of the vials.Note that the above description is generic, and some equipment designvariations are available.

The objective of a lyophilization process is to achieve a freeze-driedprotein cake with acceptable appearance, biological potency, ease ofreconstitution, and long-term storage stability. A prudently designedlyophilization cycle is one that is robust, consumes less time andenergy, and maintains product quality. Both formulation-related andcycle-related factors contribute to achieving this goal.

For freeze-dried products, the formulation and the lyophilizationprocess are intricately interrelated. As mentioned earlier, to maintainproduct stability, the product temperature needs to be below its glasstransition temperature (Tg′) both during drying and storage. Therefore,a formulation with a higher Tg′ allows drying at a higher temperaturecompared with a lower-Tg′-formulation and subsequently expedites thefreeze-drying time. Since Tg′ of the formulation is approximately themass-average of Tg′ values of all the amorphous components in theformulation, the Tg′ of the formulation can be raised by increasing theweight fraction of high-Tg′ components of the formulation and/or bydecreasing the weight fraction of low-Tg′ components. Of course, it isnecessary that the chosen excipients regardless of their Tg′ values,protect the protein from possible degradations.

The addition of a lyophilization excipient in the processes describedherein may be necessary. One or more excipients may be added. Thelyophilization excipient(s) contemplated for use in the presentprocesses include sucrose, lactose, mannitol, dextran, sucrose, heparin,glycine, glucose, glutamic acid, gelatin, sorbitol, histidine, dextrose,trehalose, methocel, hydroxy ethyl cellulose, hydroxy ethyl starch,poly(ethylene glycol), poly(vinyl pyrolidone) and polyvinyl alcohol, orvarious combinations thereof, as well as other buffers, proteinstabilizers, cryoprotectants, and cyropreservatives commonly used bythose skilled in the art. The present invention provides an improvedlyophilization and reconstitution method for concentrating PEGsTNF-R1 togreater than 45 mg/ml.

Therapeutic uses of the compositions of the present invention depend onthe biologically active agent used. One skilled in the art will readilybe able to adapt a desired biologically active agent to the presentinvention for its intended therapeutic uses. Therapeutic uses for suchagents are set forth in greater detail in the following publicationshereby incorporated by reference including drawings. Therapeutic usesinclude but are not limited to uses for proteins like consensusinterferon (see, U.S. Pat. Nos. 5,372,808, 5,541,293, herebyincorporated by reference including drawings), interleukins (see, U.S.Pat. No. 5,075,222, hereby incorporated by reference includingdrawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868,5,618,698 5,547,933, 5,621,080, 5,756,349, and 5,955,422, herebyincorporated by reference including drawings), granulocyte-colonystimulating factors (see, U.S. Pat. Nos. 4,999,291, 5,581,476,5,582,823, 4,810,643 and PCT Publication No. 94/17185, herebyincorporated by reference including drawings), megakaryocyte growthdifferentiation factor (see, PCT Publication No. 95/26746), stem cellfactor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, herebyincorporated by reference including drawings), OB protein (see PCTpublication Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816hereby incorporated by reference including figures), and novelerythropoiesis stimulating protein (PCT Publication No. 94/09257, herebyincorporated by reference including drawings). In addition, the presentcompositions may also be used for manufacture of one or more medicamentsfor treatment or amelioration of the conditions the biologically activeagent is intended to treat.

As relates specifically to PEGsTNFR1, the present invention provides formethods for the treatment of certain diseases and medical conditions(many of which can be characterized as inflammatory diseases) that aremediated by TNF. A disease or medical condition is considered to be a“TNF-mediated disease” if the spontaneous or experimental disease isassociated with elevated levels of TNF in bodily fluids or in tissuesadjacent to the focus of the disease or indication within the body.TNF-mediated diseases may also be recognized by the following twoconditions: (1) pathological findings associated with a disease can bemimicked experimentally in animals by the administration of TNF and (2)the pathology induced in experimental animal models of the disease canbe inhibited or abolished by treatment with agents which inhibit theaction of TNF. Many TNF-mediated diseases satisfy two of these threeconditions, and others will satisfy all three conditions. Anon-exclusive list of TNF-mediated diseases, as well as the relatedsequela and symptoms associated therewith, is adult respiratory distresssyndrome; cachexia/anorexia; cancer (e.g., leukemias); chronic fatiguesyndrome; congestive heart failure; graft versus host rejection;hyperalgesia; inflammatory bowel disease; neuroinflammatory diseases;ischemic/reperfusion injury, including cerebral ischemia (brain injuryas a result of trauma, epilepsy, hemorrhage or stroke, each of which maylead to neurodegeneration); diabetes (e.g., juvenile onset Type 1diabetes mellitus); multiple sclerosis; ocular diseases; pain;pancreatitis; pulmonary fibrosis; rheumatic diseases (e.g., rheumatoidarthritis, osteoarthritis, juvenile (rheumatoid) arthritis, seronegativepolyarthritis, ankylosing spondylitis, Reiter's syndrome and reactivearthritis, psoriatic arthritis, enteropathic arthritis, polymyositis,dermatomyositis, scleroderma, systemic sclerosis, vasculitis, cerebralvasculitis, Sjögren's syndrome, rheumatic fever, polychondritis andpolymyalgia rheumatica and giant cell arteritis); septic shock; sideeffects from radiation therapy; systemic lupus erythematous; temporalmandibular joint disease; thyroiditis and tissue transplantation.Specifically, TNF-mediated diseases (e.g., diseases mediated by TNF-αand/or TNF-β) may be treated by administering to a patienttherapeutically effective amounts of truncated sTNFRs or derivativesthereof.

The PEGsTNF-R1 products each may be administered to a patient intherapeutically effective amounts for the treatment of TNF-mediateddiseases, as defined above, including such as rheumatic diseases (e.g.,lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriaticarthritis, rheumatoid arthritis and staphylococcal-induced (“septic”)arthritis). The term “patient” is intended to encompass animals (e.g.,cats, dogs and horses) as well as humans.

A PEGsTNF-R1 product may be administered via topical, enteral orparenteral administration including, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal,intraventricular and intrasternal injection and infusion. A truncatedsTNFR product may also be administered via oral administration or beadministered through mucus membranes, that is, intranasally,sublingually, buccally or rectally for systemic delivery.

It is preferred that PEGsTNF-R1 products be administered viaintra-articular, subcutaneous, intramuscular or intravenous injection.Additionally, PEGsTNFR1 product may be administered by a continuousinfusion (e.g., constant or intermittent implanted or external infusionflow-modulating devices) so as to continuously provide the desired levelof PEGsTNFR1 product in the blood for the duration of theadministration. This is preferably accomplished by means of continuousinfusion via, e.g., mini-pump such as osmotic mini-pump. In these ways,one can be assured that the amount of drug is maintained at the desiredlevel and one can take blood samples and monitor the amount of drug inthe bloodstream. Various pumps are commercially available, fromsuppliers such as MiniMed Inc, Sylmar, Calif. (e.g., MT507) and AlzaCorp., Palo Alto, Calif. (e.g., Alzet osmotic pump, model 2MLI).

PEGsTNF-RI may be administered using an autoinjector type device. Thesedevices typically use a pre-filled syringe or pre-filled cartridge withthe device. The device is held against the injection site, a needleinserts through the skin and injects the drug in approximately 5-30seconds depending on the device and syringe configuration. The use ofcommercially available devices and syringes requires viscosities of <400cP for an injection to occur in a reasonable time, i.e. <30 seconds andmore preferably <15 seconds. Commercial suppliers are ScandinavianHealth Ltd. Far Eastern World Center, 11th Floor-8, #77, Hsin Tai WuRood, Sec. 1, Hsih Chih, Taipei, Taiwan, R.O.C., and Owen Mumford Ltd.Brook Hill, Woodstock, Oxford OX20 1TU, England.

It is also contemplated that other modes of continuous ornear-continuous dosing may be practiced. For example, chemicalderivatization may result in sustained release forms of the proteinwhich have the effect of continuous presence in the blood stream, inpredictable amounts based on a determined dosage regimen.

Modes of using PEGsTNF-R1 products for the treatment of TNF-mediateddiseases, including inflammatory conditions of a joint (e.g.,osteoarthritis, psoriatic arthritis and rheumatoid arthritis), are setforth in European Patent Application 567566, the teachings of which arehereby incorporated by reference. By way of example but not limitation,in one specific embodiment PEGsTNFR1 products may be administeredintra-articularly for the treatment of rheumatoid arthritis andosteoarthritis. By way of example but not limitation in another specificembodiment, PEGsTNF-R1 products may be administered subcutaneously orintramuscularly for the treatment of rheumatoid arthritis, inflammatorybowel disease, cachexia/anorexia or multiple sclerosis. By way ofexample but not limitation, in a still further specific embodimentPEGsTNF-R1 products may be administered intravenously for the treatmentof brain injury as a result of trauma, epilepsy, hemorrhage or stroke;or administered intraventricularly for the treatment of brain injury asa result of trauma. A preferred mode for the treatment of arthritisincludes: (1) a single intra-articular injection of a PEGsTNF-R1 productgiven periodically as needed to prevent or remedy the flare-up ofarthritis and (2) periodic subcutaneous injections of a PEGsTNFR1product. The initiation of treatment for septic shock should begin assoon as possible after septicemia or the chance of septicemia isdiagnosed. For example, treatment may be begun immediately followingsurgery or an accident or any other event that may carry the risk ofinitiating septic shock. Preferred modes for the treatment of adultrespiratory distress syndrome include: (1) single or multipleintratracheal administrations of a PEGsTNF-R1 product and (2) bolus orcontinuous intravenous infusion of a PEGsTNF-R1 product. Regardless ofthe manner of administration, the treatment of a TNF-mediated diseaserequires a dose or total dose regimen of a PEGsTNF-R1 effective toreduce or alleviate symptoms of the disease. Other factors indetermining the appropriate dosage can include the disease or conditionto be treated or prevented, the severity of the disease, the route ofadministration, and the age, sex and medical condition of the patient.Further refinement of the calculations necessary to determine theappropriate dosage for treatment is routinely made by those skilled inthe art, especially in light of the dosage information ad assaysdisclosed herein. The dosage can also be determined through the use ofknown assays for determining dosages used in conjunction withappropriate dose-response data. The specific dose is calculatedaccording to the approximate body weight or body surface area of thepatient.

The frequency of dosing depends on the pharmacokinetic parameters of thePEGsTNF-R1 in the formulation used. The PEGsTNF-R1 may be administeredonce, or in cases of severe and prolonged disorders, administered dailyin less frequent doses or administered with an initial bolus dosefollowed by a continuous dose or sustained delivery. When administeredparenterally, parenteral unit doses, for example, may each be up to 10mg, generally up to 15 mg and more generally up to 20 mg. Whenadministered into an articular cavity, the pharmaceutical composition ispreferably administered as a single injection from, for example, a 3 to10 ml syringe containing a dose, for example, of between about 5 mg/mlto 10 mg/ml truncated sTNFR dissolved in isotonic phosphate bufferedsaline. The preparation may be administered into an articular cavity ata frequency, for example, of once every 7 to 10 days. In such a manner,the administration is continuously conducted, for example, 4 to 5 timeswhile varying the dose if necessary.

In some cases, PEGsTNF-R1 products may be administered as an adjunct toother therapy and also with other pharmaceutical formulations suitablefor the indication being treated. A PEGsTNF-R1 product and any of one ormore traditional or new anti-inflammatory drugs may be administeredseparately or in combination.

Present treatment of TNF-mediated diseases, as defined above, includingacute and chronic inflammation such as rheumatic diseases (e.g., lymedisease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriaticarthritis, rheumatoid arthritis and staphylococcal-induced (“septic”)arthritis) includes first line drugs for control of pain andinflammation classified as non-steroidal, anti-inflammatory drugs(NSAIDs). Secondary treatments include corticosteroids, slow actingantirheumatic drugs (SAARDs) or disease modifying (DM) drugs. AdditionalTNF-mediated diseases contemplated are those described in PCT WOUS97/12244.

Preferred PEGsTNF-R1 formulations contemplated for use in the presentinvention will contain one or more buffering agents such as, but notlimited to acetate, histidine or phosphate; a tonicity modifier such as,but not limited to sucrose, sorbitol, mannitol, or glycine; anantioxidant such as, but not limited to methionine, EDTA, or ascorbate;an antimicrobial agent such as, but not limited to benzyl alcohol orphenol; a surfactant such as, but not limited to polysorbate 20 orpolysorbate 80.

It is contemplated that when sorbitol is the tonicity modifier, it isbetween zero and 5.48%, more preferably 1% to 5.48%, more preferably1.5% to 5.48%, more preferably 2% to 5.48%, and even more preferably2.56% to 5.48%. It is contemplated that when glycine is the tonicitymodifier, it is between zero and 2.19%, more preferably 1% to 2.19%,more preferably 1.25% to 2.19%, and even more preferably 1.5% to 2.19%.In one particular embodiment, the formulation comprises acetate bufferat between pH 4-5 and 2.56% Sorbitol. It is understood that the abovepercentages are based on weight/volume.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.Additional methods for reducing the solution viscosity of a PEGsTNFR1formulation may include site-directed mutagenesis of specific aminoacids or removal of amino acids sequences contained within the codingregion of the sTNFR1 amino acid sequence.

Example 1

This example describes experiments wherein PEGsTNF-RI at 45 cP and 336cP was loaded into 1 ml syringes and injected with commerciallyavailable autoinjectors. The results of these studies are shown in thefollowing table (Table 1). The standard autoinjector was modified todeliver the 336 cP solution.

TABLE 1 Viscosity (cP) Needle Size Injection time (sec) 45 26 10.5 45 2742 336 23 9 336 25 78The data demonstrate that while the autoinjector can deliver the lowviscosity solution in less than 15 sec with the 26 G syringe, asignificantly larger needle was necessary to make an equivalentinjection with the higher viscosity material. Smaller needles (26 or 27G) are preferred over the larger needles (23 G) to reduce injectionpain. This shows the importance of developing formulations of PEGsTNF-R1having sufficiently high concentrations and with sufficiently lowviscosities.

Example 2

This example describes experiments wherein various concentrated samplesof PEGsTNF-R1 were prepared and then viscosity measurements taken onconcentrated samples.

The samples for this experiment were prepared by room temperaturediafiltration at the indicated pH in 10 mM sodium acetate. The membranesused for the diafiltration were in the form of cassettes, and themembrane types were regenerated cellulose with nominal molecular weightcut-off value of 5 kD and 10 kD. The starting material enters throughthe feed port and buffer-exchanged product exits through the retentateport. Filtrate was removed from filtrate ports. Transmembrane pressure,crossflow rate, and filtrate flowrate were monitored and controlledduring the process. After concentration of the protein, 140 mM sodiumchloride (NaCl), 5.48% sorbitol, or 2.19% glycine was added andviscosity measurements taken.

Viscosity was measured using a Brookfield viscometer (BrookfieldInstruments, USA). The system was temperature stated at 16° C. using acirculating water bath. Viscosity measurements were recorded afterequilibration of the system. The results of this analysis are depictedin Table 2.

TABLE 2 pH of Final protein Viscosity Sample # sample concentrationExcipients (cP) c 5.0 59 mg/ml none 532 2 4.5 55 mg/ml NaCl (0.9%) 550 34.5 55 mg/ml Sorbitol (5.48%) 276 4 4.5 55 mg/ml Glycine (2.19%) 255 54.5 55 mg/ml NaCl (0.30%) 355 Glycine (0.723%) Sorbitol (1.81%) 6 4.5 55mg/ml Glycine (1.10%) 255 Sorbitol (2.74%) 7 5.0 55 mg/ml NaCl (0.9%)719 8 5.0 55 mg/ml Sorbitol (5.48%) 335 9 5.0 55 mg/ml Glycine (2.19%)327 10 5.0 55 mg/ml NaCl (0.30%) 544 Glycine (0.723%) Sorbitol (1.81%)11 5.0 55 mg/ml Glycine (1.10%) 326 Sorbitol (2.74%) 12 4.5 45 mg/mlNaCl (0.9%) 245 13 4.5 45 mg/ml Sorbitol (5.48%) 134 14 4.5 45 mg/mlGlycine (2.19%) 125 15 4.5 45 mg/ml NaCl (0.30%) 167 Glycine (0.723%)Sorbitol (1.81%) 16 4.5 45 mg/ml Glycine (1.10%) 126 Sorbitol (2.74%) 175.0 45 mg/ml NaCl (0.9%) 309 18 5.0 45 mg/ml Sorbitol (5.48%) 157 19 5.045 mg/ml Glycine (2.19%) 164 20 5.0 45 mg/ml NaCl (0.30%) 220 Glycine(0.723%) Sorbitol (1.81%) 21 5.0 45 mg/ml Glycine (1.10%) 154 Sorbitol(2.74%)This example demonstrates that pH, protein concentration and additionalexcipients all affect the final viscosity, and that formulations havingconcentrations of up to 55 mg/ml and viscosities <400 cP can be obtainedusing sorbitol and/or glycine as a formulation excipient.

Example 3

This example describes experiments wherein samples of PEGsTNF-R1 werelyophilized using varying pH's, protein concentrations, andlyophilization methods. The lypohilized samples were then reconstitutedto concentrations >50 mg/ml and viscosity measurements taken.

PEGsTNF-R1 at 25 mg/ml for lyophilization was prepared as follows:PEGsTNF-R1 was buffer exchanged into water, concentrated using an Amiconstirred cell device, and diluted with 10× concentrated buffer to 25mg/ml in 10 mM histidine, pH 4.0 or 5.5, 1% (w/v) sucrose, 2% (w/v)glycine and 0.01% polysorbate 20.

PEGsTNF-R1 at 60 mg/ml for lyophilization was prepared as follows:PEGsTNFR1 was buffer exchanged into water, concentrated using an Amiconstirred cell device, and diluted with 10× concentrated buffer to 25mg/ml in 10 mM histidine, pH 4.0 or 5.5, 1.0% (w/v) sucrose, 2% (w/v)glycine and 0.01% polysorbate 20.

Samples were then lyophilized using the low temperature method or hightemperature method as described in the Materials and Methods sectionbelow. After lyophilization, the samples were reconstituted with waterto the desired protein concentration and with the following excipientconcentrations: 5 mM histidine, pH 4.0 or 5.5, 0.5% (w/v) sucrose, 1%(w/v) glycine and 0.005% polysorbate 20. Viscosity was measured using aHaake falling ball microviscometer (Haake Instruments, Germany). Thesystem was temperature stated at 26° C. using a circulating water bath.Viscosity measurements were recorded after equilibration of the system.The results of this analysis are depicted in Table 3.

TABLE 3 Solution Final protein for concentration Vis- SampleLyophilization recon- after cosity # method stitution reconstitution(cP) 1 Lyophilized Low Sterile   57 mg/ml 230 at 25 mg/ml Temperaturewater and pH 4.0 method 2 Lyophilized Low Sterile   57 mg/ml 515 at 25mg/ml Temperature water and pH 5.5 method 3 Lyophilized Low Sterile 72.2mg/ml  410* at 25 mg/ml Temperature water and pH 4 method 4 LyophilizedLow 10% 72.9 mg/ml  570* at 25 mg/ml Temperature sucrose and pH 4 method5 Lyophilized Low Sterile   60 mg/ml 246 at 60 mg/ml Temperature waterand pH 4 method 6 Lyophilized High Sterile   60 mg/ml 266 at 60 mg/mlTemperature water and pH 4 method *Due to the high viscosity, sampleswere measured at 37° C.This example again demonstrates that pH, protein concentration andadditional excipients all affect the final viscosity, and thatformulations having concentrations of at least 57 mg/ml and viscosities<400 cP can be obtained using various ultrafiltration/lyophilizationtechniques.

Example 4

This example describes experiments wherein samples of PEGsTNFR1, atvarious concentrations, and containing various excipients, were testedfor stability.

Samples for stability studies of PEGsTNFR1 at concentrations of 15 mg/mlwere prepared by buffer exchanging the protein into deionized waterusing the tangential flow system described above. Excipients (e.g.,histidine, acetate) were then added from stock solutions to their finalconcentrations and pH. The samples were then sterile filtered and 1 mlaliquots filled in 3 cc glass vials and incubated at the indicatedtemperature.

Samples for stability studies of PEGsTNFR1 at concentrations 45 mg/mlwere buffer exchanged into 10 mM acetate. The pH after concentrating was4.9. Excipients were added from stock solutions to the indicatedconcentrations. The samples were then sterile filtered and 1 ml aliquotsfilled in 3 cc glass vials and incubated at the indicated temperature.

Stability of PEGsTNFR1 was determined by high performance size exclusionchromatography. A TosoHaas TSKGSW3000xL (7.8×300 mm) size exclusioncolumn was equilibrated in buffer containing 10mM sodium acetate pH 5.0,0.5M sodium chloride, 10% ethanol (v/v). Protein was eluted using a flowrate of 0.5 ml/min. The results of this analysis are depicted in Table4.

TABLE 4 Incu- % Main bation Peak by Buffer/ Tonicity Protein Temp SEC atExcipients Modifier pH (mg/ml) (° C.) 12 weeks 10 mM Acetate 140 mM NaCl4 15 4 92.9 10 mM Acetate 140 mM NaCl 5.5 15 4 93.4 10 mM Histidine 140mM NaCl 5.5 15 4 93.3 10 mM Acetate 140 mM NaCl 4 15 37 67.2 10 mMAcetate 140 mM NaCl 5.5 15 37 87.3 10 mM Histidine 140 mM NaCl 5.5 15 3782.0 10 mM Acetate NaCl (140 mM) 4.9 52 4 97.4 10 mM Acetate Sorbitol(5.48%) 4.9 45 4 97.0 10 mM Acetate Glycine (2.19%) 4.9 46 4 96.9 10 mMAcetate NaCl (140 mM) 4.9 52 37 73.1 10 mM Acetate Sorbitol (5.48%) 4.945 37 86.1 10 mM Acetate Glycine (2.19%) 4.9 46 37 88.4The data demonstrate that pH, temperature, protein concentration andchoice of excipients are all factors which affect the stability and thatstable formulations having concentrations of >45 mg/ml can be prepared.

Materials and Methods

All chemicals were ACS grade or better.

The sTNFRs used in the present invention were prepared according to theabove incorporated-by-reference PCT WO US97/12244.

The PEGsTNFR1 formulations used in the present invention were preparedusing the selective N-terminal chemical modification as described byKinstler et al. (U.S. Pat. Nos. 5,824,784 and 5,985,265).

The low temperature lyophilization of the PEGsTNF-R1 was carried out asfollows: Vials were loaded onto a shelf equilibrated at 4° C. The shelftemperature was decreased to −50° C. at a cooling rate of 36° C./hr.After holding at −50° C. for two hours, the shelf temperature wasincreased to −15° C. at a heating rate of 35° C./hr and held there fortwo hours and 30 minutes. The samples were then brought back to −50° C.at a cooling rate of −23° C./hr. The primary drying was started byevacuating the chamber to 80 mTorr and held at −50° C. for an additional30 minutes. The shelf temperature was brought to −25° C. at a heatingrate of 12.5° C./hr, then kept at −25° C. for seventeen hours. Thesecondary drying was initiated by bringing the shelf temperature to 30°C. by heating at a rate of 5.5° C./hr. After 12 hours at 30° C., thesecondary drying was complete.

The high temperature lyophilization of the PEGsTNF-R1 was carried out asfollows: Vials were loaded onto a shelf equilibrated at 4° C. The shelftemperature was decreased to −40° C. at a cooling rate of 15° C./hr.After holding at −40° C. for two hours, the shelf temperature wasincreased to −15° C. at a heating rate of 10° C./hr and held there fortwo hours. The primary drying was started by evacuating the chamber to80 mmHg. The shelf temperature was kept at −15° C. for one hour and thenincreased to 10° C. at a heating rate of 10° C./hr. The primary dryingwas continued for 30 hours at 10° C. The secondary drying was continuedby increasing the shelf temperature to 30° C. at a heating rate of 10°C./hr. After 14 hours at 30° C., the secondary drying was complete.

The present invention has been described in terms of particularembodiments found or proposed to comprise preferred modes for thepractice of the invention. It will be appreciated by those of ordinaryskill in the art that, in light of the present disclosure, numerousmodifications and changes can be made in the particular embodimentsexemplified without departing from the intended scope of the invention.

What is claimed is:
 1. A pharmaceutical formulation comprising: (a) a tonicity modifier, wherein the tonicity modifier is sorbitol and/or glycine; and (b) a pegylated erythropoietin at a concentration of at least 45 mg/mL; wherein the viscosity of the pharmaceutical formulation is less than 400 cP.
 2. The formulation of claim 1, wherein the tonicity modifier comprises 1% to 5.48% (w/v) sorbitol, preferably 1.5% to 5.48% (w/v), 2% to 5.48% (w/v), or 2.56% to 5.48% (w/v).
 3. The formulation of claim 1, wherein the tonicity modifier comprises 5.48% (w/v) sorbitol.
 4. The formulation of claim 1, wherein the tonicity modifier comprises 1% to 2.19% (w/v) glycine, preferably 1.25% to 2.19% (w/v) or 1.5% to 2.19% (w/v).
 5. The formulation of claim 1, wherein the tonicity modifier comprises 2.19% (w/v) glycine.
 6. The formulation of claim 1, wherein the tonicity modifier comprises 1.1% (w/v) glycine and 2.74% (w/v) sorbitol.
 7. The formulation of claim 1, further comprising a buffer.
 8. The formulation of claim 7, wherein the buffer is acetate.
 9. The formulation of claim 1, wherein the formulation has a pH in the range of 4.0 to 5.0.
 10. The formulation of claim 1, wherein the pegylated erythropoietin is at a concentration of up to 55 mg/mL.
 11. A pharmaceutical formulation comprising: (a) a tonicity modifier, wherein the tonicity modifier is 1% to 5.48% (w/v) sorbitol and/or 1% to 2.19% (w/v) glycine; (b) an acetate buffer; and (c) a pegylated erythropoietin at a concentration of at least 45 mg/mL up to 55 mg/mL; wherein the formulation has a pH of 4.0 to 5.0.
 12. The formulation of claim 11, wherein the pH is 4.9. 